Cytochromes c are critical for prokaryotic cellular processes such as aerobic and anaerobic respiration, photosynthesis and detoxification. Cytochromes c are unique among the cytochromes due to their requirement for the covalent attachment of the heme co-factor, a process termed cytochrome c biogenesis. In prokaryotes the process can occur via two pathways termed System I and System II. The focus of this proposal will be System II, which consists of two integral membrane proteins, CcsA and CcsB. Through recombinant expression in E. coli, our lab has recently purified a fused CcsBA protein from Helicobacter hepaticus and shown that it is a functional holocytochrome c synthetase and heme transporter. The catalytic function of CcsBA depends on its ability to interact with the heme co-factor and apocytochrome c. I have undertaken an extensive genetic analysis of the conserved residues in CcsBA to identify residues that are critical for its molecular function(s). I this proposal I aim to further probe the molecular function(s) of CcsBA through elucidation of the synthetase-apocytochrome c binding domain (Aim1) and the development of an in vitro reconstitution assay (Aim2) that will be used to probe the chemical requirements (e.g. Fe+2, reducing agents, unfolded cytochrome c) and steps in cytochrome c biogenesis (e.g. order of attachment). By taking advantage of recent successes in the recombinant system II, a mechanistic understanding of the System II holocytochrome c synthetase function will emerge. This is critical because this pathway is found in a broad range of bacteria, including the pathogens that cause tuberculosis, meningitidis, ulcers and whooping cough. This pathway is an ideal target for antimicrobials as it is required for the growth of these organisms and putative active sites of CcsBA are outside of the cell cytoplasm, similar to the targets of the penicillin class of antibiotics. These mechanistic studies will determine the factors that are critical for prokaryotic cytochrome c biogenesis, but not required for mammalian cytochrome c maturation. An additional benefit of these studies is that the technologies and cytochrome c plasmids generated can be used to unravel the mechanism of the System I pathway, present in alpha and gamma proteobacterial pathogens.